The challenge in systemic cytotoxic cancer therapy is to eradicate primary tumors and metastatic disease while sparing normal tissue from off-target effects of chemotherapy. Anthracyclines such as doxorubicin are effective chemotherapeutic agents for which dosing is limited by development of cardiotoxicity. Our published evidence shows that targeting CD47 enhances radiation-induced growth delay of tumors while remarkably protecting soft tissues. The protection of cell viability observed with CD47 is mediated autonomously by activation of protective autophagy. We examined whether CD47 blockade also protects cancer cells from cytotoxic chemotherapy. To evaluate blockade of CD47 in combination with chemotherapy in vivo, we employed the 4T1 breast cancer model and examined tumor and cardiac tissue viability as well as autophagic flux. A high-throughput screen revealed that blockade of CD47 does not interfere with the cytotoxic activity of anthracyclines against 4T1 breast cancer cells. Targeting CD47 enhanced the effect of doxorubicin chemotherapy in vivo by reducing tumor growth and metastatic spread by activation of an anti-tumor innate immune response. Moreover, systemic suppression of CD47 protected cardiac tissue viability and function in mice treated with doxorubicin. Our experiments indicate that the protective effects observed with CD47 blockade are mediated through upregulation of autophagic flux. However, the absence of CD47 in did not elicit a protective effect in cancer cells, but it enhanced macrophage-mediated cancer cell cytolysis. Therefore, the differential responses observed with CD47 blockade are due to autonomous activation of protective autophagy in normal tissue and enhancement immune cytotoxicity against cancer cells. CD47 is a ubiquitous cell surface receptor that directly regulates T cell immunity by interacting with its inhibitory ligand thrombospondin-1 and limits clearance of cells by phagocytes that express its counter-receptor signal-regulatory protein-alpha. Murine natural killer (NK) cells express higher levels of CD47 than other lymphocytes, but the role of CD47 in regulating NK cell homeostasis and immune function remains unclear. Cd47-/- mice exhibited depletion of NK precursors in bone marrow, consistent with the antiphagocytic function of CD47. In contrast, antisense CD47 knockdown or gene disruption resulted in a dose dependent accumulation of immature and mature NK cells in spleen. Mature Cd47-/- NK cells exhibited increased expression of NK effector and interferon gene signatures and an increased proliferative response to interleukin-15 in vitro. Cd47-/- mice showed no defect in their early response to acute Armstrong lymphocytic choriomeningitis virus (LCMV) infection but were moderately impaired in controlling chronic Clone-13 LCMV infection, which was associated with depletion of splenic NK cells and loss of effector cytokine and interferon response gene expression in Cd47-/- NK cells. Broad CD47-dependent differences in NK activation, survival, and exhaustion pathways were observed in NK cell transcriptional signatures in LCMV infected mice. These data identify CD47 as a cell-intrinsic and systemic regulator of NK cell homeostasis and NK cell function in responding to a viral infection. We are involved in a collaboration to identify and develop small molecule molecular probes and pretherapeutic agents that would selectively target SIRPalpha or TSP1 interactions with CD47, provide a route to optimize pharmacokinetics, reduce on-target toxicity and maximize tissue penetration, and allow more flexible routes of administration. As the first step toward this goal, we developed an automated quantitative high-throughput screening (qHTS) assay platform capable of screening large diverse drug-like chemical libraries to discover novel small molecules that inhibit CD47-SIRPalpha interaction. Using time-resolved Forster resonance energy transfer (TR-FRET) and bead-based luminescent oxygenchanneling assay formats (AlphaScreen), we developed biochemical assays, optimized their performance, and individually tested them in small-molecule library screening. Based on performance and low false positive rate, the LANCE TR-FRET assay was employed in a 90,000 compound library qHTS, while the AlphaScreen oxygen channeling assay served as a cross-validation orthogonal assay for follow-up characterization. With this multi-assay strategy, we successfully eliminated compounds that interfered with the assays and identified five compounds that inhibit the CD47-SIRPalpha interaction; these compounds will be further characterized and later disclosed. Importantly, our results validate the large library qHTS for antagonists of CD47-SIRPalpha interaction and suggest broad applicability of this approach to screen chemical libraries for other protein-protein interaction modulators.